The long term goal of our work is to develop a successful anti-malaria vaccine. The burden of malaria, with up to two million deaths per year due to Plasmodium falciparum infection, illustrates the urgency for such a vaccine. In particular, we propose herein to study the immunological basis for the protective immunity against pre-erythrocytic stage parasites that is induced by immunizations with distinct genetically attenuated parasite (GAP) in mice. Understanding the mechanisms involved in protective immunity could then provide the foundation for rational design and development of an effective malaria vaccine. Preliminary data indicate that LS parasites may be the predominant targets of protective immunity and that LS proteins also contribute to the induction of protective immunity induced by GAP vaccines. Using P. yoelii GAPs (PyGAP) as model, we demonstrated that one genetically deficient parasite [Pyuis4(-)] induced more potent and prolonged protective immunity than another [Pyuis3(-)], and that mice immunized with different GAPs recognized different antigens. Based on these data, we hypothesize that the variation in protective immunities induced by different GAPs may involve different immune mediators and a divergent profile of immune biomarkers and target antigens that distinguish protective from non-protective, and short-lived from long-lived, immunity. In this project, we propose to test our hypothesis via three aims: In aim 1, we will establish a systematic model of protective immunity following three different PyGAPs and irr-spz immunization that represent a range of protective immunity - complete long-lasting, partial and short-lived, or non-protective immunity against wild type P. yoelii sporozoite challenge. We can then characterize T cell responses (magnitude, phenotypes and proliferation) associated with the different degrees and durations of protective immunity in different trains of mice. In Aim 2, we will determine the distinctive immune biomarkers that associate with differentially protective immunity induced by different PyGAPs. Aim 3 will delineate the effector mechanisms of protective CD8 T cell responses induced by PyGAP. The outcome of this study, a better understanding of the T cell-mediated immunity, identification of immune biomarkers of protection against malaria infection, and effector mechanisms of protective immunity induced by PyGAP vaccines, will be invaluable for development of an effective subunit malaria vaccine. PUBLIC HEALTH RELEVANCE: Despite decades of research, there is still an urgent need for development of a successful anti-malaria vaccine. We have shown that Genetically Attenuated Parasites (GAP) can confer long term protective immunity against malaria. In the study proposed herein, we aim to elucidate and characterize, at the molecular and cellular levels, the mechanisms involved with such protective immunity. Results of this project will then provide the foundation for development of an effective anti-malaria vaccine.